Neglected tropical disease (NTD) diagnostics: current development and operations to advance control

ABSTRACT

Neglected tropical diseases (NTDs) have become important public health threats that require multi-faceted control interventions. As late treatment and management of NTDs contribute significantly to the associated burdens, early diagnosis becomes an important component for surveillance and planning effective interventions. This review identifies common NTDs and highlights the progress in the development of diagnostics for these NTDs. Leveraging existing technologies to improve NTD diagnosis and improving current operational approaches for deployment of developed diagnostics are crucial to achieving the 2030 NTD elimination target. Point-of-care NTD (POC-NTD) diagnostic tools are recommended preferred diagnostic options in resource-constrained areas for mapping risk zones and monitoring treatment efficacy. However, few are currently available commercially. Technical training of remote health care workers on the use of POC-NTD diagnostics, and training of health workers on the psychosocial consequences of these diagnostics are critical in harnessing POC-NTD diagnostic potential. While the COVID-19 pandemic has challenged the possibility of achieving NTD elimination in 2030 due to the disruption of healthcare services and dwindling financial support for NTDs, the possible contribution of NTDs in exacerbating COVID-19 pandemic should motivate NTD health system strengthening.

1. Introduction

Prior to the COVID-19 pandemic and consequent global recession, sub-Saharan Africa (SSA) experienced tremendous economic growth and included some of the world’s fastest-growing economies [1]. However, economic growth rates varied significantly across African countries because of the availability of resources, conflict, and poor governance [2]. These, among other factors, are to blame for the region’s challenges to eliminate certain infectious diseases, particularly neglected tropical diseases (NTDs).

The term ‘NTD’ was coined in Berlin, Germany in 2003 during a stakeholder meeting to set the tone for global NTD initiatives [3]. NTD is currently defined using various criteria focused on its characteristics. Generally, the term encompasses a diverse group of diseases or conditions caused by a variety of pathogens that affect over 2.7 billion people worldwide, primarily in the poorest populations living in low to middle-income countries (LIMC) of Africa, Asia, and Latin America [4,5]. According to the World Health Organization (WHO), NTDs include ‘Buruli ulcer, Chagas disease, dengue and chikungunya, dracunculiasis (Guinea-worm disease), echinococcosis, foodborne trematodiases, human African trypanosomiasis (sleeping sickness), leishmaniasis, leprosy (Hansen’s disease), lymphatic filariasis, mycetoma, chromoblastomycosis and other deep mycoses, onchocerciasis (river blindness), podoconiosis, rabies, scabies and other ectoparasitoses, schistosomiasis, soil-transmitted helminthiases, snakebite envenoming, taeniasis/cysticercosis, trachoma, and yaws and other endemic treponematoses’ [6]. Sub-Saharan Africa (SSA) accounts for up to 90% of the global NTD-associated burden, and this has been linked to poverty and climatic conditions that promote the transmission of some of these NTDs [5].

Climate change can affect the spread of NTDs by altering environmental conditions. Temperature impacts the reproduction, survival, and distribution of pathogen-carrying vectors and their hosts, as well as pathogen replication. Rainfall determines the suitability of habitats for vectors and hosts, affecting their breeding sites [7,8]. Additionally, climate change consequences such as drought-induced food and water insecurity, along with conflict-driven population displacement, worsen the impact of climate change on NTDs in resource-limited areas of sub-Saharan African countries [9]. Several SSA countries are currently addressing the issue of NTD by scaling up mass drug administration (MDA) programmes; for example, in Nigeria and Kenya, where 15 or more of the 20 NTDs listed by the WHO respectively, are either suspected, confirmed, or endemic [10,11].

NTDs interfere with productivity in all economic sectors, causing affected countries to lose billions of dollars yearly and further complicating the lives of impoverished people who live on less than $2 per day [12]. NTDs also impair school-aged children’s cognitive development and significantly impact school attendance, limiting the educational opportunities of children in the affected countries [2]. NTDs’ ability to trap individuals in a vicious cycle of poverty, and social stigma at the family and community levels [13], makes them diseases of major public health concern.

Accurate diagnosis of affected populations is critical for rational control and many endemic countries have recently demonstrated a renewed commitment to NTD control programmes. While NTD control programmes are usually geared toward MDA, diagnosis deficiency in control programs has been identified as one of the roadblocks to effective control [10]. In fact, diagnosis is important to realize the 2030 elimination target as it is a key component of NTD mapping, screening, surveillance, monitoring, and evaluation [14,15]. Currently, the diagnosis of NTDs involves various methods such as microscopy, molecular techniques, serological tests, or point-of-care (POC) tests [16]. The selection of diagnostic tools for low-resource settings, where NTDs are prevalent, takes into account factors like performance, operational ease, and cost considerations. Microscopy is laborious and time-consuming; many molecular and serological tests are not cost-effective and will require technical expertise. In NTD-endemic low-resource areas, there is a push to prioritize and promote the use of POC diagnostic tools. However, challenges related to regulatory procedures, pricing and funding, implementation issues, and quality assurance hinder the widespread adoption of POC diagnostics in these regions [16].

NTDs and COVID-19 exhibit various interconnected relationships. For instance, schistosomiasis has been indicated to elevate COVID-19 case fatality rates in endemic countries in Africa compared to non-endemic regions [17]. Increasing treatment coverage for NTDs could potentially decrease active COVID-19 cases and enhance the rate of recovery [17]. The implementation of MDA in NTD-endemic areas was affected by COVID-19 due to disruptions in health systems caused by the pandemic [18]. Additionally, the pressing demand to develop diagnostics for COVID-19 has resulted in the suspension of the development of rapid diagnostic tests (RDTs) and molecular tests for numerous tropical diseases, including NTDs, although this challenge could at the same time present opportunities for diagnostic development for NTDs by adopting COVID-19 strategies [19]. To address NTDs diagnostic challenges, the WHO established a Diagnostic Technical Advisory Group (DTAG) as part of the latest NTD roadmap. Its main objectives are to tackle key aspects of NTD diagnostics, identify gaps in access to existing tools, and provide guidance on necessary innovations and developments. The aim is to ensure that the diagnostic process effectively informs decision-making in the efforts to combat NTDs [20]. This review will highlight some of the various NTD diagnostic approaches that have already been developed or are in the process of being developed, as well as emerging or new-generation NTD diagnostics, and operational approaches for deploying these diagnostics in order to meet the 2030 NTD elimination target. Importantly, we shall focus on a few of the NTDs in the Diagnostics Technical Advisory Group (DTAG) prioritization of diagnostic needs in endemic countries.

2. Neglected tropical diseases control strategies

2.1. Preventive chemotherapy

The WHO currently recognizes 20 NTDs [8], five of which (onchocerciasis, lymphatic filariasis (LF), soil-transmitted helminthiasis (STHs), schistosomiasis, and trachoma) can be managed with preventive chemotherapy (PC). These are the so-called PC-NTDs [21]. Preventive chemotherapy can be given to the entire at-risk population (mass drug administration; MDA) or to a specific at-risk group (targeted treatment) to treat individuals with the infection, prevent the development of new morbidity, or stop disease transmission [3]. Almost all of the countries in the WHO Africa Region have one or more of these NTDs that can be managed with PC, and 17 of these countries have five of these diseases co-endemic [8]. African regions together with other WHO regions endemic for NTDs i.e. Asia and the Americas reported the treatment of 732 million individuals for at least one of the PC-NTDs in 2020 [22]. To meet the public health targets for PC-NTD, effective and sustained control programs require repeated rounds of drug delivery to endemic areas over several years, with high therapeutic and geographic coverage [21]. The availability of PC in NTD-endemic areas is heavily reliant on community drug distributors (CDDs), who distribute drugs within their communities [8].

MDA is an important approach in NTD programming, but its success is frequently hampered by the difficulty of achieving universal access and coverage for effective and long-term public health impact. According to reports, a significant number of at-risk groups are not adequately represented in NTD programming. In 2019, for example, there were 0.8, 3.7, and 37.6 million people in Benin, Kenya, and Nigeria respectively, who did not receive NTD treatment [23]. The current NTD national program master plans for endemic countries in many SSA countries have yet to address the challenges of equity in NTD program planning, implementation, and evaluation.

2.2. Vector control

Vector control is critical for the prevention and control of disease transmission, and it has been widely advocated for vector-borne disease prevention and control. Because most NTDs are transmitted by vectors (insects) or intermediate hosts (such as aquatic snails), the same NTD prevention and control approach is strongly recommended [24].

Unlike malaria and yellow fever vector control efforts, vector control for onchocerciasis, Chagas disease, human African trypanosomiasis (HAT), leishmaniasis, and lymphatic filariasis (LF) is rarely prioritized, particularly in SSA countries [25]. LF (Wuchereria bancrofti) is one example of NTD spread by mosquito species (Anopheles, Aedes, Culex, Mansonia, and Ochlerotatus). Although a common interventional control program may be adequate to eliminate LF vectors, much emphasis is placed on the use of MDA to eliminate microfilariae from infected people’s blood in order to interrupt the life cycle of the parasite and transmission by the mosquito vector [25].

During the Onchocerciasis Control Programme (OCP) in Africa from 1974 to 2002, vector control became an important part of the onchocerciasis control strategy [26]. From 1976 to 1989, a weekly aerial larviciding program was implemented along the rivers of a large area of the West African savanna (700,000 km²) where onchocerciasis was endemic [27]. Several African countries where the Simulium (blackfly) population is high were targeted. These include Benin, Burkina Faso, northern Côte d’Ivoire, Ghana, southern Mali, southwestern Niger, Togo, Guinea, and Sierra Leone [27]. The program’s success was confirmed by a rapid and significant decrease in the endemicity of onchocerciasis. Further evidence of the program’s effectiveness was a three to nine-fold reduction in the annual transmission potential of infective Onchocerca volvulus larvae in Burkina Faso within ten years of implementation [28]. Transmission has been reduced or eliminated in Burkina Faso since then, even in the absence of vector control or ivermectin MDA [25]. The OCP approaches have important lessons that can be applied to other vector-borne NTDs. These include the use of a targeted approach to eliminate vector larvae populations based on entomological and epidemiological monitoring data, as well as insecticide rotation in the event of resistance to mainstream insecticides [28]. The African Program for Onchocerciasis Control (APOC) was established in 1995 in 19 African countries that were not part of the OCP [29]. Although APOC countries strongly advocated for and successfully implemented ground larviciding, there was a heavy reliance on community-directed ivermectin treatment, particularly after the Merck Mectizan donation scheme began in 1987 [30].

Vector control has been successfully integrated into other control programs, for example in the control of leishmaniasis, especially in areas where sand flies are endophilic [31,32], resulting in a reduction in transmission and disease incidence. In Bangladesh, India, and Nepal, indoor residual spraying (IRS) was combined with screening and MDA, resulting in a drop in visceral leishmaniasis (VL) cases from 77,000 in 1992 to fewer than 6,000 in 2016 [33]. Although insecticide-treated nets (ITNs) did not appear to be effective against anthroponotic VL in India and Nepal [34], successful bed net deployment reduced anthroponotic cutaneous leishmaniasis (CL) incidence in a number of Old World foci [35].

2.3. Other vector management strategies

Deltamethrin impregnated in dog collars reduced zoonotic VL as well as provided high individual protection against canine infection [36]. When implemented at the community level, human infection incidence was reduced by 43 percent [37], and infantile clinical VL was reduced by 50 percent [36]. Tsetse traps and targets, as well as the incorporation of synthetic pyrethroids, have also been used to combat HAT [38]. Glossina palpalis gambiense and G. tachinoides populations were rapidly reduced in Burkina Faso in the 1970s following the deployment of deltamethrin-impregnated biconical traps [39]. Other trap variants and methods have since been developed and deployed [40]. Another approach was to use pyrethroid to protect reservoir hosts (cattle) from tsetse and tick bites, which was incorporated into control programs in Burkina Faso, Tanzania, Zanzibar, and Zimbabwe [41]. Although the use of sterile insects was only tested experimentally in Nigeria and Tanzania, the approach was programmatically adopted in only Zanzibar. It was instrumental in the country’s tsetse eradication between 1994 and 1997 [42]. Unsustainable efforts in tsetse control campaigns, combined with a neglect of surveillance activities, have since resulted in an increase in the incidence of HAT in many endemic countries.

Control of intermediate hosts of schistosomes that cause intestinal and urogenital schistosomiasis has not been implemented in areas of the highest endemicity. The decline in capacity development in malacology in endemic countries could be a contributory factor to the non-implementation of snail control. Also, the idea of mollusciciding water bodies that serve as transmission foci for schistosomiasis and the sole sources of water supplies may not be favored by communities as they often represent the poorest segment of the population without alternative water supplies. Transmission control through the eradication of intermediate snail hosts was highly prioritized in schistosomiasis control implementation programs where schistosomiasis control has been largely successful [43]. Field application of niclosamide and black plastic film coverage of water surface are among the national schistosomiasis elimination programs in China that have significantly aided the country in its fight against schistosomiasis [44]. In Japan, total eradication was achieved by integrating snail control into the country’s prevention campaigns such as toilet reform policy and education at all levels. Reinfection was completely stopped by turning waterways into concrete canals to destroy the intermediate snail host habitats [45]. It is, however, important to note that the destruction of intermediate host habitats in schistosomiasis control can have a long-term effect on the balance of natural water ecosystems. The proliferation of the gastropod hosts in water bodies, as well as the discovery of potential snail hosts in African endemic areas [46,47], highlight the need to increase efforts in snail control in schistosomiasis control in SSA.

Considering the significance of vector control in reducing the transmission of certain NTDs, it is important to address the environmental impact associated with the use of larvicides and insecticides [48]. One example highlighting this concern is the utilization of larvicidal oil for mosquito control, which was found to pose ecological risks in the coastal marine ecosystem. This oil was reported to inhibit the growth of two microalgal species in the coastal seawaters of Hong Kong [48]. Furthermore, the application of permethrin in Guinea rivers was observed to result in alterations in the community structures of invertebrates. This alteration exerted pressure on certain taxonomic groups and led to a decrease in the abundance of certain taxa [49]. In addition, laboratory evaluations of potential blackfly larvicides such as permethrin, cyphenothrin, pyraclofos, and carbosulfan demonstrated their toxicity to African fish species [50]. These instances, along with numerous others, have highlighted the necessity for the development of environmentally friendly insecticides and molluscicides that possess selective action while remaining nontoxic to non-targeted organisms or biocontrol agents [51,52].

2.4. Water, sanitation, hygiene (WASH), and education

Many of the mainstay NTD control strategies are periodic, population-based deworming (MDA), which are shown to be safe, scalable, and cost-effective [53]. Notwithstanding the benefits of MDA programs in endemic areas, the sole implementation of MDA has not provided a long-term solution [53]. This challenge necessitated the development of a comprehensive strategy for the control and prevention of worm infections by the WHO and UNICEF about two decades ago which included the provision of safe water supply and adequate sanitation as a necessary control strategy [54]. The integration of WASH with education and deworming, collectively termed WASHED, was intended to interrupt STH reinfection [55]. Further to this, the NTD road map 2021–2030 launched in 2021, in a bid to strengthen the programmatic response to NTDs includes ‘achieving universal access to at least basic water supply, sanitation, and hygiene in areas endemic for NTDs by 2030’, and calls for strengthened coordination and collaboration with WASH stakeholders to ensure that services are delivered and sustained in communities that are most affected by NTDs [10].

A notable example of the success of WASH and education in eradicating NTDs was recorded in the dracunculiasis eradication program which adopted a comprehensive approach that integrated water filtration, case management, education, and interventions to protect and improve drinking water sources [56]. The 2022 total reported cases of dracunculiasis were thirteen in five sub-Saharan African countries where cases were detected in the year [57]. WASH intervention program integrated with education also produced a reduction in the transmission of childhood intestinal parasitic infections in Ethiopia, howbeit, the reduction was not significant compared with baseline infection levels [58]. The strengthening of local health offices’ WASH education program, community mobilization to construct WASH facilities, and support for the community’s sustenance of households’ WASH performance were recommended as key to maintaining a continuous reduction in transmission [58]. While health education was believed to reduce the prevalence of active Trachoma and the intensity of S. mansoni [59,60], the paucity of data that relate WASH to the two infections makes it difficult to appreciate the impact of WASH on the transmission of the two infections.

The significance of WASH interventions in reducing the burden of NTDs cannot be overstated, and there has been a notable increase in the demand for the integration of different programs. The lack of progress in successful integration can be attributed to several common obstacles, such as divergent program objectives, excessive focus on MDA, funding disparities and isolated funding, as well as insufficient sharing of information [61]. To foster intersectoral partnerships, it is crucial to establish shared goals through educational advocacy, involve governments and ministries, develop integrated strategies, cultivate dedicated partnerships, and create a more conducive donor environment for integrated funding [61].

2.5. Diagnosis

Infectious disease diagnosis is critical for rational control. The availability of diagnostic tools has contributed significantly to the success of control and elimination of many NTDs. The Global Program to Eliminate Lymphatic Filariasis (GPELF) is regarded as one of the most effective public health responses because the treatment of affected populations is guided by a RDT that identifies LF risk zones [62]. Over 7.7 billion treatments have been administered to over 910 million people since the inception of GPELF over two decades ago [9]. This has significantly reduced the prevalence, severity, and duration of microfilaria and elephantiasis-related morbidities. Fourteen countries have now been declared LF-free, and no treatment is required in ten more [9]. The availability of improved RDT for the diagnosis of LF in many regions of eastern Africa and the Indian subcontinent has increased access to diagnosis. The RDT is easier compared to diagnosis by identification of microfilariae in blood or skin samples, antigen detection, radiographic imaging, or PCR [57]. In endemic countries, the use of RDTs as monitoring tools in high-risk areas has facilitated early treatment and improved patient outcomes toward control and elimination [62]. The development of new diagnostics such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) for Buruli ulcer, a debilitating necrotic infection, would improve early diagnosis and treatment outcomes for patients, as well as map control activities [62,63]. While RPA and LAMP may not align perfectly with the concept of POC tests due to their reliance on traditional DNA extraction and the potential duration of 30–90 min to yield results (depending on the reaction), they represent a significant stride forward in the ongoing development of POC technology [64]. There are currently unmet needs for RDT development for disease monitoring and post-elimination surveillance of many NTDs.

There are no commercially available POC tests for taeniasis cysticercosis, chromoblastomycosis, mycetoma, HAT (rhodesiense), dracunculiasis, and leprosy [65], hence, efforts in this direction are critical to leverage the current progress in their control.

2.6. Vaccines

Vaccination is widely recognized as a highly cost-effective health intervention for both human and animal populations [66]. However, despite the acknowledgment that preventive chemotherapy alone cannot achieve elimination targets for many NTDs, there are currently no licensed vaccines available for NTDs except for rabies. Only candidate vaccines for dengue virus, which are in advanced clinical development in Phase II and III, show promising progress [67]. Various anthelminthic vaccines targeting hookworm, STH infections, schistosomiasis, and onchocerciasis are in different stages of development. In Gabon, a combination of recombinant human hookworm vaccines called Na-GST-1 and Na-APR-1 is undergoing Phase I clinical testing in endemic areas [68]. Vaccine antigens for ascariasis and trichuriasis are currently undergoing preclinical testing [69]. Progress has also been made in the development of vaccines for both urogenital and intestinal schistosomiasis. These vaccines are either in Phase I trials, advanced preclinical development stages, or later stages of clinical development [70,71]. The recent human challenge model for schistosomiasis has paved the way for fast-tracked product development for the treatment and prevention of schistosomiasis [72].

Several candidate vaccines for both visceral and cutaneous forms of leishmaniasis are being developed by various organizations such as the Infectious Diseases Research Institute (IDRI) Product Development Partnership (PDP), the Sabin PDP, and two European consortia [73–75]. Furthermore, a therapeutic vaccine for Chagas disease is being developed through collaboration between the Sabin PDP and the Carlos Slim Foundation [76]. Additionally, other approaches to combat Chagas disease are being pursued in parallel [77,78].

Despite evidence demonstrating the cost-effectiveness or potential cost-savings of NTD vaccines [79,80], international agencies like WHO and Gavi have been slow to promote their adoption. This reluctance stems from factors such as limited involvement of major pharmaceutical companies and the perception that NTD vaccines primarily prevent disability rather than childhood mortality [67]. Additionally, the absence of surrogate markers for protection raises concerns about the high costs associated with advanced clinical development. To mitigate these challenges, exploring human challenge models for rapid vaccine efficacy assessment and combining NTD vaccines with those targeting malaria or other priority diseases could reduce investment risks [67].

3. Progress in diagnostic development for NTD

Accurate, reliable, and affordable diagnostic tools play a crucial role in NTD programs. They are indispensable for making informed decisions regarding individual-level treatments, guiding population-level choices such as adjusting treatment frequency or ceasing mass treatment, facilitating disease surveillance, and providing confidence in confirming the elimination or eradication of diseases. In essence, diagnostic tools drive monitoring and evaluation efforts and are vital for documenting the impact of interventions [15]. While conventional clinical and microscopic techniques are often sufficient for mapping disease distribution and monitoring progress in most NTD interventions, the importance of enhanced diagnostics becomes more evident as infection prevalence decreases and the possibility of elimination or eradication arises [81,82]. Unfortunately, the lack of dedicated funding for the advancement and improvement of diagnostic tools may have put the achievement of success in addressing these diseases at risk [15].

The WHO, Bill and Melinda Gates Foundation (BMGF), and several pharmaceutical companies endorsed the London Declaration of 2012 to support the eradication of Guinea worm disease, the elimination of LF, leprosy, HAT, and blinding trachoma; and the control of schistosomiasis, STHs, Chagas disease, VL, and onchocerciasis by 2020 [23]. Unfortunately, these targets were not met, consequently, a new road map with the target set for 2030 has been established [10].

The new road map prioritized diagnosis and identified it as one of four key areas to be strengthened in order to meet the 2030 NTD elimination target. Experience with disease elimination and eradication programs has shown that adequate surveillance tools such as diagnostics are critical success factors [83].

Guinea worm disease does not require a diagnostic test because the clinical features are clear [84]. Other common NTDs, such as schistosomiasis, STH, LF, and onchocerciasis, are diagnosed by microscopic examination of urine or stool, night blood, or skin snips, with the exception of trachoma, which is diagnosed clinically [83]. When the intensity of parasite infection is high, microscopy can be a useful diagnostic technique; however, it requires trained personnel and robust infrastructure such as electricity, and a laboratory with a functioning microscope. Microscopy becomes more laborious in populations with low parasite burdens because several rounds of diagnosis are frequently required to improve sensitivity; this is practically impossible in population-wide surveys for mapping risk zones and monitoring treatment efficacy. As elimination targets are approached, large numbers of samples must be tested to ensure that transmission has been interrupted, necessitating the use of high-throughput tests [83]. The negative impact of low intensity (more than low prevalence) is on negative predictive value and the increased probability for false-negative results due to less-than-optimal sensitivity of tests.

Antigen-based diagnostic methods, such as rapid immunochromatographic tests (ICT) or enzyme-linked immunoassays (ELISAs), may provide an alternative means of diagnosis. ICTs are simple to use and appropriate for on-site diagnosis of NTDs in the community, but they are often challenged by low sensitivity in areas with low infection [85]. ELISA, which detects antibodies directed against infections is more sensitive than microscopy but may not be suitable to differentiate between active and previous infections [77]. Multiplex and LAMP PCRs for mobile use may be used to monitor the few cases of infection that remain after treatment. The latter can also be used to monitor drug resistance in the population [86]. Routine use of PCR in SSA for this purpose is however unlikely due to cost inefficiency and limited availability of personnel with the requisite skills and training.

To address the limited availability of diagnostic tools, the WHO has created a specialized working group called the Diagnostics and Technical Advisory Group (DTAG). The DTAG’s primary function is to identify and prioritize diagnostic needs and then develop target product profiles (TPPs) for upcoming diagnostic tools [11,87]. These TPPs outline the minimal and optimal requirements for various diagnostic needs, such as simplicity, performance, and price. They are tailored to specific use-cases for NTDs, including starting, monitoring, and evaluating program performance or verifying the interruption of disease transmission [88]. The WHO has already published TPPs for lymphatic filariasis, onchocerciasis, STH, and schistosomiasis, with TPPs for other NTDs currently in development [89–91]. Nonetheless, the process of turning newly discovered diagnostic biomarkers into cost-effective laboratory or POC for endemic programs is lengthy, expensive, and uncertain [88]. The challenges of diagnosis of selected NTDs are discussed in the following

section.

3.1. Onchocerciasis

Onchocerca volvulus infection in vectors has been tracked using molecular tools [83]. Furthermore, the detection of antibodies to the O. volvulus specific antigen (Ov16) in children by ELISA was used to certify the cessation of transmission in South America and Uganda [92]. Other identified Onchocerca antigens used for serological diagnosis that have shown promising diagnostic potential with sensitivity ranging from 90–100 % are Ov31, Ov-Far-1, Ov-API-1, and Ov33-GST [93]. The cost of reagents required to process samples for ELISA, the need for power supply, trained personnel, and transportation of samples in good condition into the laboratory equipped with specialized equipment makes this method operationally difficult in resource-limited areas. The emergence of antibody-based RDT has introduced a cost-effective and field-appropriate method for onchocerciasis. Ov16 antigen was successfully transformed into a rapid format card test, achieving a sensitivity of 90.6% [94]. However, the production of this card test was discontinued in 2000, despite its promising performance in the field. Nevertheless, there has been a resurgence of interest in the Ov16 rapid test, leading to the development of two commercially available RDTs that incorporate the Ov16 antigen. These tests include a standalone IgG4 rapid test and a combined test that combines the Ov16 antigen with the W. bancrofti antigen Wb123 for dual diagnostic purposes [95].

The absence of infection in both humans and Simulium, the vector host, determine the certification of onchocerciasis elimination. To be specific, before an area can be certified free of onchocerciasis, the vector must not carry infective larvae (or be nearly free of them), and infection in sentinel populations such as children in endemic areas must be less than 0.1 percent [96]. To address this requirement, there has been a demand for the advancement of PCR-based detection methods for Onchocerca in both humans and Simulium flies. One such method, known as 0–150 PCR, has been developed to overcome the challenges of time and cost associated with traditional techniques involving skin snip examination in humans and fly dissection in Simulium [97]. A large proportion of the human population and vector hosts must be tested, and the diagnostics must have a high negative predictive value [83].

3.2. Lymphatic filariasis (LF)

Antigen-detecting ICT is preferable to antibody detection for LF because the latter is incapable of distinguishing between passive and active infections as in the case of many antibody-based diagnostic assays. ICT performance is comparable to microscopy examination of microfilariae in blood but showed lower sensitivity when compared with ELISA in low prevalence settings [98]. Lymphatic filariasis is one of the NTDs in which diagnosis is integrated into control programs. In many endemic countries, the results of diagnoses are used to guide policy toward LF control. ICT was developed to track active LF transmission and the success of MDA in endemic areas. The recombinant antigen-based antibody assays using BmR1, Bm14, and WbSXP displayed high sensitivity of > 90% in the diagnosis and surveillance of LF caused by W. bancrofti and Brugia malayi [99]. An antibody-based diagnostic tool utilizing BmR1 as the captured antigen was employed to monitor the effectiveness of MDA on B. malayi transmission. Both ICT and BmR1 demonstrated efficacy as diagnostic tools and practical approaches for determining whether to continue MDA in 6–7-year-old children [100]. An important concern arises when considering Loa loa as the primary contributing factor to the cross-reactivity observed in LF ICT. There is a substantial connection between LF ICT positivity and loiasis, evident at both individual and endemicity levels. This raised concerns regarding the accuracy of the whole blood ICT employed for LF mapping in areas where loiasis is co-endemic [101]. These assays can be adapted to a bead or microfluidics-based assay to allow for multiplexing for improved diagnostic potential [83]. ICTs designed for rapid testing of onchocerciasis and LF are commercially accessible; however, the ease of accessibility and cost have restricted their availability in remote regions characterized by the high endemicity of these diseases. Even in cases where these tests are obtainable, the affected population segment may still lack the financial means to afford them.

3.3. Schistosomiasi

Schistosomiasis, particularly urogenital schistosomiasis, is one of the most easily diagnosed NTDs as alternative diagnostic methods, which are less technically demanding than traditional microscopy, can indirectly identify the infection. In endemic areas, visible hematuria is usually used as the first line of diagnosis. This method is sometimes used in endemic areas, particularly when resources are scarce. Nonetheless, a significant portion of the population with a high infection burden may remain undetected, as hematuria is occasionally absent in such cases [102]. Although chemical reagent strips capable of detecting microscopic blood (microhematuria) and protein (proteinuria) in urine can be used to identify communities in need of MDA, they are not appropriate for low-intensity populations as the parasitic load might not be substantial enough to cause bladder damage [103]. Antigens from various stages of S. mansoni with diagnostic potential for antibody detection have been reported to be suitable for schistosomiasis monitoring in low-endemic areas with low infection intensity [104]. However, these tests are unable to distinguish between current and previous infections [105]. In low transmission circumstances, a POC-ICT designed to identify the circulating cathodic antigen (CCA) of S. mansoni has also shown superior sensitivity to Kato-Katz smears [106]. A circulating anodic antigen (CAA) for S. haematobium is currently not available commercially. A dipstick dye immunoassay was also developed and validated for fast screening of S. japonicum infection in low-endemic locations [107]. Fung et al. [108] developed a PCR test that can detect S. japonicum infection at an intensity as low as 0.5 eggs per gram of feces. A recent study looked at the use of recombinase polymerase amplification (RPA), a rapid, portable, POC molecular-based technique for detecting S. haematobium that yielded promising results [109]. However, none of these tests have been adopted for routine epidemiological use.

3.4. Soil-transmitted helminths

Soil-transmitted helminths (STHs) like Ascaris lumbricoides, Trichuris trichiura, Strongyloides stercoralis, Necator americanus, Ancylostoma duodenale, and Ancylostoma ceylanicum are common causes of NTDs [110]. These infections affect approximately 895 million people globally, resulting in a disease burden of 1.9 million disability-adjusted life years. STHs disproportionately impact individuals in rural and remote areas of low and middle-income countries [111]. Decisions regarding the implementation or discontinuation of STH control programs rely on epidemiological surveys that determine infection prevalence and intensity [110]. The Kato-Katz microscopy technique is frequently used in these surveys due to its simplicity and low resource requirements, as recommended by the World Health Organization (WHO) [112]. Nevertheless, this method has significant limitations, including poor sensitivity, the need for quick sample processing to prevent hookworm egg degradation, and the inability to differentiate between the three hookworm species or identify Strongyloides spp. infection [112,113].

According to Knopp et al. [114], the utilization of a single preparation using the FLOTAC technique exhibited considerably greater sensitivity in diagnosing low-intensity STH infection compared to three replicates of the Kato-Katz method. As a result, this approach shows promising potential for improving patient management, monitoring STH transmission, and assessing control measures at the population level [114]. While FLOTAC demonstrates high sensitivity in detecting low-intensity STH infections, it requires significantly more time for preparation compared to Kato-Katz [115]. Additionally, in terms of equipment, FLOTAC is more expensive than the Kato Katz technique and time consuming since multiple slides would have to be examined in order to obtain reliable results, as highlighted by Knopp et al. [116]. However, it is important to acknowledge that repeated stool sampling can reduce patient compliance and increase overall costs [114]. In order to address the challenges associated with implementing FLOTAC and enhance the accuracy of copromicroscopic diagnosis, a simplified device called the mini-FLOTAC was created. This method offers several notable benefits, with one key advantage being its ease of transport and suitability for laboratories with limited resources, as it eliminates the need for a centrifugation step [117]. The McMaster method, which demonstrates similar diagnostic performance to Kato-Katz, presents itself as an excellent alternative approach for monitoring extensive treatment programs. It is a reliable and precise method, with a robust multiplication factor and the ability to deliver accurate efficacy results. Furthermore, the McMaster method lends itself to easy standardization, enhancing its practicality and effectiveness [115].

Certain studies have documented the utilization of ELISA assays to capture coproantigens. These assays operate on the fundamental principle of employing rabbit anti-excretory/secretory (E/S) polyclonal antibodies to capture proteins released by the parasites [118,119]. These techniques have proven effective in diagnosing S. stercoralis and hookworm infections. Nevertheless, unlike the case with other parasites like Plasmodium species and various protozoans, methods based on antigen detection have not gained widespread usage in diagnosing STH infections.

The quantitative real-time polymerase chain reaction (qPCR) technique is a sensitive molecular diagnostic test that can identify common species of STHs in preserved stool samples [113]. It is effective for diagnosing STH infections and measuring infection intensity [112,120,121] but is mainly used in large-scale surveys, clinical trials, and STH transmission evaluation [122–124]. When dealing with diverse STH species, qPCR is more reliable than Kato-Katz in providing detailed STH burden information needed for interventions [110].

Using qPCR-derived cycle threshold (Ct) values to measure infection intensity presents certain challenges because the WHO-recommended thresholds for infection intensity were established based on measurements derived from the Kato-Katz technique. Although some studies have measured STH infection intensity using Ct values [110,112,121], additional research is required to validate this approach and standardize the evaluation of infection intensity using qPCR. Additionally, some practical issues must be considered when deciding whether to use qPCR, such as its higher perceived cost and the need for specialized equipment and trained personnel compared to microscopy [125].

Several studies have proposed the use of multiplex real-time PCR and digital multiplex PCR (dmPCR) for the simultaneous detection of parasites, offering high sensitivity and specificity [114,126]. However, the adoption of these advanced techniques like other molecular-based methods is hindered by the need for expensive reagents and instruments to interpret the results. Consequently, these methods are not suitable for developing countries where STH infections are endemic. In such contexts, conventional multiplex PCR emerges as a simple and cost-effective alternative for detecting mixed infections within a single reaction. Several multiplex PCR assays have been developed for detecting STHs, demonstrating sensitivity that is five times greater than the formalin – ethyl acetate concentration technique (FECT) for the detection of multiple infections. Additionally, these assays exhibit twice the sensitivity in detecting of S. stercoralis [86]. Significant advancements have been made in molecular diagnostics, which involve the utilization of RDTs and smart optical devices to detect various parasites, such as those causing malaria. These developments have shown remarkable progress in enhancing POC diagnosis [127,128]. However, it seems that this approach has not been fully utilized for the diagnosis of STHs.

3.5. Human African trypanosomiasis (HAT)

Because microscopic inspection of blood films is insufficient for diagnosing Trypanosoma brucei gambiense infection, the card agglutination test (CATT) is employed to identify infected individuals. A sensitive CATT has a low titer of positive results but is not specific [129]. The high probability of false positive results is only suggested in positive microscopy results [83]. Other techniques used to improve the sensitivity of microscopy include buffy coat examination after blood centrifugation and the small anion exchange column technique [77]. These options, however, are not available or feasible in resource-constrained settings where HAT is common. Conflicting results are always a challenge for HAT management, so repeat testing is advised. If the disease progresses to the central nervous system (CNS), a lumbar puncture or cerebrospinal fluid (CSF) examination is recommended, but this is not always culturally acceptable [130].

The diagnosis of HAT presents significant challenges. It is increasingly evident that conventional parasitological methods are inadequate in detecting T. b. gambiense infections among individuals who are seropositive but asymptomatic. These individuals exhibit the ability to control the infection at low levels or harbor parasites outside the bloodstream, particularly in the skin, without detectable parasitemia [131]. This poses a twofold concern: firstly, they could potentially contribute to disease transmission, and secondly, they may progress to develop clinical HAT [131,132]. Therefore, there is a critical need for specific and highly sensitive diagnostic tools that can be used at the POC and/or in a high-throughput manner in low-income countries [133].

Lateral flow ICT devices are harnessed to create RDTs that specifically identify anti-trypanosome antibodies in human blood samples obtained through finger-pricks. These RDT-based lateral flow devices are user-friendly, provide easy-to-read results, and possess stability characteristics that enable widespread distribution and availability in remote areas where the disease is prevalent [134]. Currently, the first RDT for the diagnosis of HAT developed by Standard Diagnostics (SD BIOLINE HAT) is now available commercially. The device employs native surface glycoproteins (VSG) LiTat 1.3 to detect anti-trypanosome antibodies [134]. This test demonstrates favorable levels of sensitivity and specificity in comparison to CATT [135]; however, further enhancements are still required, particularly in terms of facilitating test production and reducing costs [134].

Promising outcomes have been observed in the development of multiple molecular amplification tests for gambiense HAT. These tests include 18SrDNA-PCR, TBR-PCR, Tb177bp-qPCR, 18SrDNA-qPCR, SLRNA RT-qPCR, 18S RNA RT-qPCR, RIME-LAMP, and 7SL-sRNA RT-qPCR [136,137]. However, the widespread implementation of these tests for mass screening is constrained by cost implications and the need for adequate infrastructure.

3.6. Visceral leishmaniasis (VL)

The diagnosis of visceral leishmaniasis (VL) has traditionally relied on invasive methods such as examination of bone marrow and splenic aspirate with sensitivity of 60–85% and > 95%, respectively [138]. Although sensitive and specific, these methods are associated with risks and discomfort for patients [83]. The sensitivity of peripheral blood smears is lower (51–53%), particularly in individuals with a low parasite count in their blood [139]. While these methods are cost-effective, they necessitate skilled technicians, laboratory facilities, blood transfusion capabilities, nursing supervision, and surgical interventions. However, due to the severe adverse effects associated with such approaches, researchers are actively seeking sampling methods that are less painful, patient-friendly, and more effective [140]. To enhance the sensitivity, one approach is to inoculate the clinical sample into a culture medium such as Schneider’s insect medium or diphasic culture medium supplemented with 5–10% fetal bovine serum [141]. The enriched media provide vector-like environmental conditions that facilitate the transformation of amastigotes into promastigotes. The promastigotes can then be cultured for 7–21 days by incubating the parasite culture at a temperature of 24–26 ℃ in an incubator [140]. However, this method is time-consuming and impractical for field surveillance purposes.

Immunological assays, including the direct agglutination test (DAT), ELISA, and ICT, play a crucial role in the diagnosis of leishmaniasis, as highlighted by studies conducted by Elmahallawy et al. [139] and Singh and Sundar [142]. Among these assays, the ICT stands out due to its rapidity and accessibility, offering a strip test that requires minimal analysis time (10–15 min). However, similar to DAT, the ICT lacks the ability to differentiate between a current infection and a clinical relapse [140]. While a POC diagnostic tool that detects antibodies specific for the rk39 protein (a cloned antigen of Leishmania chagasi predictive of active VL) is available, it faces challenges such as the inability to distinguish active from previous infection and the detection of antibodies in healthy individuals, as is the case with other antibody-based immunoassays [143]. In recent studies, rKE16 has been identified as an important diagnostic biomarker with high sensitivity (92.8–100 %) and specificity (96.0–100 %). However, the sensitivity was found to be lower when compared to panels from Brazil and East Africa [144].

Several molecular-based assays, including conventional PCR, nested PCR, semi-nested PCR, real-time PCR (RT-PCR), and multiplex PCR, have demonstrated sensitivities ranging from 90.0–100% and specificities ranging from 83.0–100%. While nested PCR offers increased sensitivity and specificity compared to conventional PCR and reduces nonspecific binding, it is a non-quantitative and costly approach [140]. Additionally, it elevates the risk of contamination when PCR tubes are opened to set up the second PCR. RT-PCR and multiplex PCR, on the other hand, reduce the risk of contamination and allow for the amplification of multiple targets simultaneously using multiple primers [145]. However, nucleic acid sequence-based amplification PCR, despite having a sensitivity of 60% to 95%, cannot differentiate between different parasite species [146].

4. Current technological advancement in NTD diagnostics development

Although many of the NTD diagnostics currently available are suitable for conducting pre-elimination surveys in areas with high endemicity, their ability to facilitate post-elimination surveillance and evaluate the success of MDA programs may be limited. Biomarker-based NTD diagnostics are particularly vulnerable to the challenge of cross-reactivity, which can reduce their diagnostic sensitivity and accuracy. In LF-endemic areas, for instance, the Alere Filariasis Test Strip (FTS) is recommended by the WHO to detect circulating filarial antigens in human blood. However, the FTS is cross-reactive with L. loa antigens [147], limiting its utility for post-elimination surveillance in (previously) co-endemic regions.

The need for next-generation NTD diagnostics to address current diagnostic challenges cannot be overstated. Some of the new technologies being developed include modifications to currently used immunological and molecular methods to compensate for their deficiencies. For instance, a miniaturized microfluidic platform has been employed to replicate the ELISA immunoassay, providing a quick and low-cost alternative to the traditional ELISA [148]. In addition, bead-based immunoassay uses laser or fluorescence instead of enzymatic substrate application to detect the binding of the secondary antibody to the antigen or antibody target. This approach provides a quantitative measure of fluorescence intensities that can be used to plan community interventions. By targeting multiple pathogen antigenic epitopes, this assay increases sensitivity [149]. Another promising avenue of investigation involves the development of immunodiagnostics capable of diagnosing co-endemic NTDs in a given area. This strategy leverages cross-reactivity as a diagnostic advantage, resulting in cost-effective multiplex diagnostic tools.

NTD diagnosis has seen advancements with the transformation of immunoassays into biosensor tools. An illustrative instance is the utilization of an amperometric immunosensor to detect T. cruzi in human serum. In this approach, T. cruzi shed-acute-phase-antigen (SAPA) was affixed onto a screen-printed carbon electrode (SPCE) that was modified with gold nanoparticles (MPA-AuNP) [150]. The immunosensor, known as SAPA – MPA–AuNP/SPCE, exhibited a limit of detection of 3.03 ng mL⁻¹, displaying favorable stability and reproducibility. Notably, the method offered a quicker testing time (26 min) compared to the conventional ELISA (90 min). Given the simplicity of the sensor’s modification and immobilization techniques, it serves as a viable option for diagnosing Chagas disease. Electrochemical biosensors developed for Leishmania showcased innovative systems compared to devices designed for other NTDs. Numerous studies employed established electrochemical techniques to optimize and validate their sensors. However, Perinoto et al. [151] introduced chemometrics using principal component analysis (PCA) for detecting anti-L. amazonensis IgGs. The outcomes revealed distinct clusters that differentiated positive and negative samples, including anti-T.cruzi antibodies.

In addition to the development of bead-based immunoassays, a more accurate diagnosis can be achieved using bead-based molecular assays. These assays can detect active infections and are particularly useful in areas with low parasite intensity or for monitoring post-treatment success. Currently, a new generation of molecular diagnostics based on microfluidics and magnetism is being developed as lab-on-a-chip tools with nanowires and quantum dot barcodes [152]. These tools are user-friendly and require little technical knowledge, enabling quick diagnosis.

Nucleic acid amplification tests (NAATs) have emerged as a preferred diagnostic approach in recent years, especially for NTDs, due to their high diagnostic sensitivity which makes them the gold standard for other diagnostic approaches. The development of LAMP assays that can be converted into POC-NAATs has allowed for the diagnosis of VL using blood samples and post-kala-azar dermal leishmaniasis (PKDL) using a skin biopsy [149]. In the case of HAT, the LAMP assay demonstrated a sensitivity and specificity of 87.3% (95% confidence CI 80.9–91.8%) and 92.8% (95% confidence CI 86.4–96.3%), respectively, when compared to a PCR gold standard. Isothermal amplification reactions share an equivalent potential for specificity as PCR, with LAMP, in particular, exhibiting enhanced specificity owing to the presence of multiple primer pairs [153]. Moreover, the combination of smartphones and nanotechnologies holds great potential for the development of low-cost multiplex DNA hybridization assays that can transmit data to a central database. This approach could provide real-time surveillance data from the field [154]. By combining LAMP technology with a handheld portable device (SMART-LAMP), which performs real-time isothermal nucleic acid amplification reactions based on colorimetric measurements, a recent study validated the diagnostic usefulness of the device in different infectious diseases, such as schistosomiasis and strongyloidiasis [155]. The developed SMART-LAMP device is Bluetooth-controlled by a dedicated smartphone app, and the study showed that the combination of long-term stabilized LAMP master mixes, which are stored and transported at room temperature, with the SMART-LAMP device provides an improvement toward true POC diagnosis of infectious diseases, particularly in settings with limited infrastructure [155].

New Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based systems are being developed to enhance specificity downstream of isothermal NAATs since these methods may have relatively low specificity [65]. The combination of the CRISPR-based diagnostic system known as SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) with the integration of RPA (Recombinase Polymerase Amplification) and CRISPR-Cas13a detection has showcased its capacity to effectively detect S. japonicum and S. mansoni [156]. This innovative approach allows for the use of either fluorescent or colorimetric indicators to assess the results. These findings underscore the potential of the SHERLOCK/CRISPR-based diagnostic platform to deliver highly precise and readily deployable point-of-care tests. Such tests hold great promise as the next generation of diagnostic and surveillance tools for parasitic neglected tropical diseases [156]. In the study by Cai et al. [157], the droplet digital PCR (ddPCR) exhibited superior performance in detecting S. japonicum fecal DNA compared to the traditional Kato-Katz (KK) stool microscopy and ELISA, with sera ddPCR serving as the reference standard. Moreover, the fecal ddPCR method demonstrated a sensitivity of 98.1% (95% confidence CI 93.5–99.8%) in contrast to ELISA-based methods that employed parasite recombinant antigens (sensitivity range: 59.3–91.7 %), with KK as the diagnostic reference standard. The costly nature of ddPCR assays presents a significant obstacle to their implementation in screening campaigns, similar to many other molecular diagnostics [158]. Additionally, the requirement for specialized and advanced equipment and the need for trained personnel further complicates the deployment of molecular methods in the field [157].

Using next-generation sequencing (NGS)-generated sequence data and the Galaxy-based RepeatExplorer computational pipeline, the genomes of each organism for highly repetitive, non-coding DNA elements are searched in order to identify diagnostic targets capable of providing optimal limits of detection and species-specificity of detection [159]. Using these targets to design small-volume, multi-parallel tests, a platform that provides cost-minimizing implementation of only those assays appropriate for a specific geographic region based upon the infections present was created [160]. This approach has been used to identify high copy-number repeats for a series of STH species responsible for the greatest burden of disease in sub-Saharan Africa. Using these repetitive sequences as targets in the design of novel real-time PCR assays, the limits of detection and species-specificity of detection were improved against an established PCR-based diagnostic methodology [161].

By utilizing next-generation sequencing (NGS) and the RepeatExplorer computational pipeline, researchers are able to search for highly repetitive, non-coding DNA elements in the genomes of various organisms. This enables the identification of diagnostic targets with optimal limits of detection and species-specificity [160]. A platform was developed using these targets, allowing for the design of small-volume, multi-parallel tests. This approach minimizes costs by implementing only those assays suitable for a specific geographic region based on the prevalent infections in that area [160]. This methodology has been employed to identify high copy-number repeats in several STH species, which are responsible for a significant disease burden in sub-Saharan Africa. The repetitive sequences identified are then utilized as targets in the design of novel real-time PCR assays, resulting in improved limits of detection and species-specificity compared to established PCR-based diagnostic methods [161].

Metabolomics, a technique that utilizes high-resolution mass spectrometry, enables the identification of a broad range of metabolic species [162]. By analyzing the abundance of small metabolites (<1200 Da) in the biofluids of infected individuals, differences can be detected to discriminate between various disease states, ultimately leading to the development of novel diagnostic tools [163]. Studies have focused on investigating potential metabolite biomarkers in patients with NTDs such as HAT, schistosomiasis, and onchocerciasis [164–166].

To address the fact that most NTD programs rely heavily on microscopic examination of slides, an automated slide scanning system combined with artificial intelligence (AI) was developed and deployed in Cambodia, Ethiopia, Kenya, and Tanzania. The system created an image database for STH and S. mansoni which causes intestinal schistosomiasis. The AI model showed high average weighted precision (94.9 ± 0.8%) and weighted average recall (96.1 ± 2.1%) for all STH species, while it recorded lower precision (91.8%) and recall (86.2%) for S. mansoni. The lower performance of the device in diagnosing intestinal schistosomiasis was mainly attributed to the limited size of the training data collected [88]. These techniques have the potential to close the diagnostic gap for NTDs by 2030, but their practical implementation will require significant commitments from all stakeholders involved in NTD programming.

5. NTD-POC diagnosis and operational challenges

A point of care (POC) test is defined by Bharadwaj et al. [65] as ’a specific and sensitive assessment in which a user must administer a minimum number of steps to obtain an easy-to-interpret, rapid [within a short turnaround time (TAT)], and robust result.’ It is used when a health problem requires quick diagnosis and does not need sophisticated equipment or routine laboratory procedures.

POC tests have the potential to be an effective initial diagnostic option prior to considering more invasive tests. Although several POC diagnostics are currently being developed for many NTDs, only a few have undergone regulatory assessment and quality assurance testing.

The United States Food and Drug Administration (FDA) maintains a catalog of approved tests. Among the NTD-POC diagnostics, 16 of them are based on immunological reactions, while the remaining 7 are nucleic acid-based [65]. Immunological-based NTD-POC diagnostics, including those used to detect schistosomiasis, often face challenges in low-endemic areas due to their sub-optimal performance. Over the past two years, it has been demonstrated that commercially available POC-CCA for S. mansoni lacks inter-batch reproducibility [167]. Moreover, POC-CCA has shown an unacceptably low specificity in pregnant women and infants under nine months, as evidenced by studies conducted by Casacuberta-Partal et al. [168] and Graeff-Teixeira et al. [169]. The absence of uniformity in the development of LAMP-based POC diagnostic devices has resulted in an intricate manufacturing process and heightened production complexity. Additionally, when transitioning laboratory molecular assays into POC tests, a significant obstacle was the requirement to first extract nucleic acid as a preliminary step in sample preparation, followed by performing the LAMP reaction [155]. These challenges underscore the need for either creating new dependable diagnostic tools or optimizing existing ones [85,170,171].

The implementation requirements for NTD-POC diagnostics vary depending on the disease and context. For example, in cases of co-infections, quality assessment and field validation of pilot tests, field-deployable sample preparation, and confirmatory test (test of cure) are critical [172], but for Chagas, a field-deployable nucleic acid-based test to detect congenital Chagas disease is a critical implementation need since repeated diagnosis may be necessary to screen out false positive results in non-infected infants that carry low maternal DNA from infected mothers [173].

The use of available NTD-POC tests is heavily reliant on NTD awareness. Inadequate awareness and a lack of test kits in endemic areas were identified as factors that frequently limit the effective use of POC tests [65]. Poor analytical quality or sample handling can lead to inaccurate tests, necessitating the need for personnel training in proper test protocols, result documentation, and device maintenance. Furthermore, clinicians are concerned about the clinical relevance of POC tests in comparison to conventional reference diagnostic standards [65]. These concerns have frequently led to medical practitioners in remote areas mistrusting even sophisticated POC tests [174].

The inability of remote health workers to provide adequate posttest counseling may have a significant impact on the mental and social well-being of patients suffering from HAT, Chagas disease, leprosy, CL, Buruli ulcer, or mycetoma. As a result, in addition to providing a reliable POC test and adequate treatment options, awareness and counseling among local health workers are required to provide psychosocial support [65]. Furthermore, because NTD screening test is frequently based on physical examination and suspicion in regions of high disease endemicity in the region, a negative POC test result can challenge and jeopardize the relationship between the patient and the healthcare worker or medical practitioner [175]. This could potentially lead to a greater general mistrust of Western medicine and a shift in healthcare-seeking behavior toward traditional healers [65]. Other challenges are associated with frequent under-equipping of health services in endemic regions to manage NTD and a lack of government support for the programs. These can make it difficult to implement innovative NTD-POC tests, especially when new POC tests do not confer a direct economic advantage over existing laboratory-based diagnostic tests in the region [176]. Common POC tools or assays for some NTDs are presented in Table 1.

Table 1.

POC diagnostics of some neglected tropical parasitic diseases and their performances.

NTD	Type of PoC	Specimen type	Endemicity characteristics	Countries implemented	Diagnostic performance	Reference
Schistosomiasis	Sm – CCA	Urine	Low or potentially endemic area	Brazil	Specificity; 62.1 %	Graeff-Teixeira et al. [169]
	Sm – CCA	Urine	Low	Philippine	Sensitivity; 29.6 % Specificity; 93.8 %	Cai et al. [176]
	Sm – CCA	Urine	Moderate-high	Côte d’Ivoire, Ethiopia, Uganda, Zambia	Sensitivity; 67.0–99.1 % Specificity; 6.0–100 %	Cavalcanti et al. [177]
	Sh – SchistoScope	Urine	Endemic	Ghana and Côte d’Ivoire	Sensitivity; 91.0 % Specificity; ≥ 91.0 %	Armstrong et al. [178]
	Sh – phone-based microscope	Urine	Endemic	Ghana	Sensitivity; 72.1 % Specificity; 100 %	Bogoch et al. [179]
	Sh – ICTs	Sera	Endemic	Gabon, Tanzania, and Zimbabwe	Sensitivity; 75–89 % Specificity; 100 %	Vengesai et al. [180]
	Sm – CAA	Urine	Low endemic	Brazil	Sensitivity; 64.0 % Specificity; 73 %	Sousa et al. [181]
	Sh – UCP-LF CAA	Urine	Low endemic	Zanzibar, Tanzania	Sensitivity; 65.5–96.6 %	Knopp et al. [182]
	Sh – RPA	Urine	High endemic	Pemba island, Zanzibar	Sensitivity; 93.7 % Specificity; 100 %	Archer et al. [183]
	Sh – RPA (FGS)	Cervicovaginal lavage and vaginal self-swab samples	High endemic	Zambia	Sensitivity; 64.4–93.3 % Specificity; 91.8–98.1 %	Archer et al. [184]
	Sh – LAMP	Urine	High endemic	Angola	Sensitivity; 86.7 % Specificity; 100 %	Gandasegui et al. [185]
	Sh – LAMP	Urine	High endemic	Ghana	Sensitivity; 100 % Specificity; 100 %	Lodh et al. [186]
	Sm – LAMP	Urine	High endemic	Zambia	Sensitivity; 100 % Specificity; 100 %	Price et al. [187]
	Sm – LAMP	Fecal	High endemic	Kenya	Sensitivity; 97 % Specificity; 100 %	Mwangi et al. [188]
STH	Al – LAMP	Fecal	High endemic	Kenya	Sensitivity; 96.3 % Specificity; 61.5 %	Shiraho et al. [189]
	FC/FOB (Al, Hw, Tt, Ss)	Fecal	High endemic	Côte d’Ivoire, Lao PDR and Pemba Island, Tanzania	NA	Patel et al. [190]
	Mobile Microscope (Xiaomi PocophoneF1, Bq Aquaris X2) (Tt)	Fecal	High endemic	Kenya	Precision; 95.2–100 % Recall ; 66.2–93.6 %	Dacal et al. [191]
	USB Video Class (UVC) microscope (Al)	Fecal	Endemic	Madagascar	Sensitivity; 82.9 % Specificity; 97.1 %	Yang et al. [192]
	Ss – ICT	Sera	NA	Blood bank – Faculty of Medicine, Khon Kaen University, Thailand	Sensitivity; 96.3 % Specificity; 83.7 %	Sadaow et al. [193]
	Ss – ICT	Sera	NA	Migrants from sub-Saharan Africa in Italy	Sensitivity; 82.4 % Specificity; 73.8 %	Tamarozzi et al. [194]
	Ss – SMART – LAMP	NA	NA	NA	NA	García-Bernalt Diego et al. [155]
Onchocerciasis	Ov16 RDT	Blood	Endemic	Cameroon	NA	Ekanya et al. [195]
	OV16 RDT	Sera	Endemic	Congo	Sensitivity; 74.8 % Specificity; 98.6 %	Hotterbeekx et al. [196]
Loasis	Loa – LAMP	Blood	NA	Equatorial Guinea	Sensitivity; >9.0 % Specificity; near 100 %	Ta-Tang et al. [197]
	CellScope Loa	Blood	Endemic	Cameroon	Sensitivity; 100 % Specificity; near 94 %	D’Ambrosio et al. [198]
Lymphatic filariasis	Filariasis Test Strip (FTS)	Blood	High	Democratic Republic of the Congo (DRC)	Sensitivity; 89.8–96 % Specificity; 8.0–92 %	Chesnais et al. [199]
	Lf – LAMP	Blood	High endemic	Kenya	Sensitivity; 89.8–96 % Specificity; 8.0–92 %	Kinyatta et al. [200]
	Semi-automated microfluidic device; miniPCR-duplex lateral flow dipstick	Blood	NA	Thailand	NA	Phuakrod et al. [201]
Leishmaniasis	Vl – LAMP	Blood	NA	Brazil	Sensitivity; 98.2 % Specificity; 98.1 %	de Avelar et al. [202]
	Cl – LAMP	Tissue biopsy		Columbia	Sensitivity; 98.2 % Specificity; 98.1 %	Adams et al. [203]
	VL – Lateral flow-based rK28/39 rapid tests	Sera	Endemic	Sudan, Bangladesh	Sensitivity; 95.9–98.1 % Specificity; 86.3–88.7 %	Pattabhi et al. [204]
	Cl – Detect™ Rapid Test (CL Detect)	Tissue biopsy	Endemic	Suriname	Sensitivity; 35.8–36.7 % Specificity; 83.3–85.7 %	Schallig et al. [205]
	Loopamp™ Leishmania Detection Kit (Loopamp)	Tissue biopsy	Endemic	Suriname	Sensitivity; 84.8–91.4 % Specificity; 42.9–91.7 %	Schallig et al. [205]
	Cl Detect™ Rapid Test (CL Detect)	Tissue biopsy	Endemic	Afghanistan	Sensitivity; 65.4 % Specificity; 100 %	Vink et al. [206]
	Loopamp™ Leishmania Detection Kit (Loopamp)	Tissue biopsy	Endemic	Afghanistan	Sensitivity; 65.4 % Specificity; 100 %	Vink et al. [206]
HAT	Dual-Antigen Lateral Flow Test (Tbg)	Sera	NA	WHO Biobank – Chad, Guinea, Congo DRC, Tanzania, Uganda, Malawi	Sensitivity; 97.3–99.3 % Specificity; 46.0–83.3 %	Sullivan et al. [207]
	Dual-Antigen Lateral Flow Test (Tbr)	Sera	NA	WHO Biobank – Chad, Guinea, Congo DRC, Tanzania, Uganda, Malawi	Sensitivity; 58.9–83.9 % Specificity; 44.9–71.7 %	Sullivan et al. [207]
	Inkjet printer-produced dried LAMP (CZC-LAMP HAT – Tbg)	Blood	NA	Malawi	Sensitivity; 72.0 %	Hayashida et al. [208]
	Inkjet printer-produced dried LAMP (CZC-LAMP HAT – Tbr)	Blood	NA	Malawi	Sensitivity; 8.0%	Hayashida et al. [208]
	SHERLOCK4HAT: A CRISPR-based tool kit – Tgb	Dried blood spot, whole blood, buffy coat	NA	WHO specimen Biobank	Sensitivity; 26.5–56.1 % Specificity; 88.7–98.4 %	Sima et al. [133]
	SHERLOCK4HAT: A CRISPR-based tool kit – Tgb	Dried blood spot, whole blood, buffy coat	NA	WHO specimen Biobank	Sensitivity; 79.0–100 % Specificity; 94.1–100 %	Sima et al. [133]

Abbreviations Urine-based up-converting phosphor-lateral flow circulating anodic antigen – UCP-LF CAA; Recombinase polymerase amplification – RPA; fecal calprotectin – FC, fecal occult blood – FOB; Loop-Mediated Isothermal Amplification – LAMP; immunochromatographic test – ICT, rapid diagnostic test – RDT Schistosoma mansoni – Sm; Schistosoma haematobium – Sh; Ascaris lumbricoides – Al; Strongyloides stercoralis – Ss; Strongyloides venezuelensis – Sv; Hookworm – Hw; Onchocerca volvulus – Ov; Loa loa – Loa; Trypanosoma brucei gambiense – Tbg; Trypanosoma brucei rhodiense – Tbr Lymphatic filariasis – Lf; Visceral leishmaniasis – Vl; Cutaneous leishmaniasis – Cl; Human African trypanosomiasis – HAT; Specific High-sensitivity Enzymatic Reporter unlocking – SHERLOCK; Clustered Regularly Interspaced Short Palindromic Repeats – CRISPR; Not available – NA.

6. Financing and support for NTD diagnostics

Partnerships such as Product Development Partners (PDPs) and ‘Access public-private partnerships (PPPs)’ offer a promising avenue for achieving the elimination of NTDs [207]. Currently, strategies for controlling and eliminating NTDs have primarily relied on MDA, where pharmaceutical companies donate drugs that are repeatedly administered to populations [209,210]. This approach, known as ‘preventive chemotherapy,’ as designated by the WHO, has been effective in interrupting disease transmission and achieving elimination for diseases such as lymphatic filariasis through the Global Program to Eliminate Lymphatic Filariasis, and trachoma through the International Trachoma Initiative [211]. However, despite the acknowledged need for reliable and affordable NTD diagnostics, most PDPs primarily prioritize the development of medical products like drugs and vaccines rather than diagnostics [212]. This underinvestment in NTD diagnostics has been identified by the WHO as a significant gap in achieving the global targets for NTD elimination by 2030. To address this issue, the WHO’s Diagnostic Technical Advisory Group for Neglected Tropical Diseases (DTAG-NTD) was established in 2019 with the purpose of providing guidance on the development of diagnostic tools for this sector [20].

There has been a decrease in funding for NTD research in the past 10 years [213]. The amount of investment in NTD control currently stands at US$341 million, which equates to 23 cents per infected individual [213]. Despite the critical role of research in the development and deployment of effective diagnosis, individual case management, community-directed treatment, and surveillance, only a small portion of the funding is earmarked and disbursed for research, development, and procurement of RDTs for NTDs. The financial challenges posed by the COVID-19 pandemic introduced additional complications exacerbating the insufficient funding with further reductions in 2020 and 2021 from the diversion of funds to pandemic control [19]. For example, some funds programmed for NTDs were completely canceled by some agencies such as the Korea International Cooperation Agency, whose Global Disease Eradication Fund recently canceled plans for new funding set to begin in 2021. This fund was to be derived from an air ticket solidarity levy system. The global reduction in air travels as a result of the pandemic adversely affected the generation of resources for the fund [214].

RDT development and manufacturing phases include prototype development and technical validation, manufacturing validation, performance evaluation and clinical validation, endorsement, and scale-up, all of which require funding [215]. This is especially difficult for NTD RDTs, which are produced at a high cost, have a limited number of manufacturers, and experience frequent stock-outs. These shortcomings limit access to and use of RDTs when they are required [216]. Passive screening with RDTs can be performed using existing healthcare facilities for mapping preventive chemotherapy and delivering drugs that would otherwise be given in MDA programs, as is done for HAT and VL [19,216,217].

7. Addressing implementation challenges in the effective control of NTD

Modern technology has revolutionized the field of NTD diagnostics, enhancing diagnostic capacity in unprecedented ways. Cutting-edge tools such as molecular assays, point-of-care devices, and mobile health applications have significantly improved the accuracy, speed, and accessibility of NTD diagnosis. Molecular techniques like PCR and next-generation sequencing (NGS) enable the detection of NTD pathogens with high sensitivity and specificity. Point-of-care devices, such as RDTs and portable molecular platforms, bring diagnostics to resource-limited settings, enabling real-time detection and immediate treatment decisions. Additionally, mobile health applications provide remote diagnostic support, data management, and surveillance capabilities. Expanding efforts to successfully conclude clinical trials for the most promising NTDs diagnostics and ensuring their commercial availability will significantly contribute to closing the diagnostic gap. By giving priority to the funding of NTD diagnostics and adopting the advanced market commitment model, where donors commit in advance to purchasing a successful product at a predetermined price and quantity, accessibility can be greatly improved [218].

Local communities play a pivotal role in combating NTDs due to their intimate knowledge of the affected areas, cultural practices, and social dynamics. Their involvement and active participation are crucial for the success of any intervention or program. They act as agents of change by raising awareness about NTDs, dispelling myths and misconceptions, and promoting preventive measures within their communities. They can effectively educate their members about the importance of hygiene, sanitation, vector control, and early detection, empowering individuals to take charge of their own health [219]. Furthermore, local communities can serve as advocates, voicing their concerns and needs to policymakers and healthcare providers. By engaging in dialogue with relevant stakeholders, they can ensure that NTD control programs are tailored to the specific needs of their communities, addressing cultural, economic, and infrastructural challenges that may hinder effective interventions with respect to diagnosis of NTDs and other interventions. Empowering local communities and involving them in decision-making processes fosters a sense of ownership, leading to sustainable solutions and long-term impact [219,220].

By strengthening the healthcare infrastructure, improving access to quality healthcare services, and enhancing surveillance and reporting systems, health systems can effectively prevent, diagnose, and treat NTDs. Capacity building of healthcare workers, community engagement, and integrated approaches that address multiple NTDs simultaneously are essential components of health system strengthening. Moreover, sustainable financing mechanisms, strong leadership, and collaboration between various stakeholders are vital for the successful control [221] of NTDs. By prioritizing health system strengthening, countries can establish resilient healthcare systems capable of providing comprehensive NTD control programs and ensuring the long-term success in combating these debilitating diseases.

Developing approaches and strategies to optimize the effectiveness of available diagnostic, chemotherapy, and vector management approaches can mitigate some of the challenges NTD programs may face. Several stakeholders including the Global Network for Neglected Tropical Diseases, the BMGF, and the WHO among others, have raised the profile of operational research and more recently implementation research for NTDs. These research domains can provide avenues to address implementation barriers and bottlenecks inhibiting success in NTD programs such as; health workers and community confidence in diagnostic tools, schedules of MDA out of sync with the communities’ schedules, poor or nonexistent feedback loops to provide program results to community drug distributors and communities; delays in delivering drugs to and from national stores to primary health care centers, refusal of individuals and groups to take the NTD treatments; insufficient time and funds for promotional and educational messages, inadequate numbers and overburdened health personnel, reduction in the number of people suffering from NTD in the community making risk less apparent and leading to complacency among health workers and the community and sustainability of control efforts by national programs [222,223].

By sharing knowledge, resources, and best practices, countries can collaborate to implement effective prevention, treatment, and surveillance strategies. Coordinated efforts, funding partnerships, and joint research initiatives are essential for the global control and elimination of NTDs, ensuring that no country is left behind in the fight against these debilitating diseases.

8. Future directions

The future direction in the field of NTD diagnostics should address several key aspects to advance control and improve outcomes. These include:

1. Innovation and technological advancements: Embracing emerging technologies, such as point-of-care devices, molecular diagnostics, and digital health solutions, to enhance the accuracy, speed, and accessibility of NTD diagnostics. Investing in research and development to create novel diagnostic tools tailored to specific NTDs and addressing the unique challenges of each disease.

2. Integration and multi-disease approaches: Promoting integrated diagnostic approaches that enable simultaneous testing for multiple NTDs. This approach can streamline diagnostic processes, reduce costs, and facilitate targeted treatment interventions.

3. Capacity building and training: Strengthening diagnostic capabilities at the community and healthcare provider levels through comprehensive training programs. Empowering local healthcare workers to effectively utilize and interpret diagnostic tools in order to improve case detection and management.

4. Collaboration and partnerships: Fostering collaboration among researchers, healthcare providers, policymakers, and funding agencies to promote knowledge sharing, resource mobilization, and joint efforts in NTD diagnostics. Establishing partnerships between academia, industry, and governments to accelerate diagnostic development and scale-up.

5. Access and equity: Ensuring equitable access to NTD diagnostics, particularly in underserved and marginalized communities. Address barriers such as cost, infrastructure limitations, and geographical challenges to ensure that diagnostics reach those in need.

6. Data and surveillance: Strengthening NTD surveillance systems by integrating diagnostic data into comprehensive disease surveillance platforms. Leverage data analytics and digital tools to enhance monitoring, early detection, and response to NTDs.

7. Policy and funding support: Advocacy for policy frameworks that prioritize NTD diagnostics, including funding allocation, regulatory guidelines, and procurement strategies. Encouraging governments and international organizations to invest in diagnostics as a crucial component of NTD control programs.

Conclusions

NTDs frequently coexist in endemic areas; thus, control strategies for closely related NTDs can be combined and integrated. This can also be used in operational approaches for diagnostics deployment for mapping high-risk zones for NTDs and monitoring treatment efficacy. There is a need for more efficient diagnostics for NTDs, particularly those that have low sensitivity, and efforts should be doubled for others that currently lack diagnostics. NTDs have been shown to have a negative impact on COVID-19 outcomes [43], so while pandemics should be prioritized, NTDs should not be overlooked. In addition to technical training for remote health workers on the use of POC-NTD diagnostic kits, training health workers on the psychosocial impact of POC diagnosis results and implementation research to address limitations to the effective deployment of tools and strategies and program sustainability remains critical.

Funding Statement

The author(s) reported there is no funding associated with the work featured in this article.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

OTO and OO – conceptualization and contents design; OTO – wrote the manuscript’s first draft; OO, MS, LMS, and RGF – critical revision of manuscript draft; all authors approved the final manuscript version.